Unlike normal cells which primarily generate ATP by coupling glycolysis to oxidative phosphorylation through the tricarboxylic acid (TCA) cycle, cancer cells often alter their metabolism and enhance glycolysis to support tumor growth (1-3). Cancer cells with mitochondrial defects or mutations in TCA cycle enzymes, encounter challenges in generating the metabolic precursors needed for cell proliferation. To compensate for this, these cells reverse the direction of the TCA cycle to generate citrate and other metabolites from glutamine in a process known as glutamine-dependent reductive carboxylation (GDRC) (4, 5). As a recently discovered form of metabolism, very little is known about the regulation of this pathway in different types of cancers. Our laboratory recently identified a subset of lung cancer cell lines which undergo GDRC. Differential gene expression analysis (6) revealed that high GDRC cell lines possess an epithelial phenotype, characterized by high E-cadherin expression and low vimentin expression, compared with low GDRC cell lines which exhibit a mesenchymal phenotype. Bioinformatic analyses (7) revealed that high GDRC cell lines often harbor mutations in the epidermal growth factor (EGF) receptor and exhibit sensitivity to EGFR inhibitors, including Erlotinib and Gefitinib. The studies outlined in this proposal are designed to learn more about the regulation of GDRC and whether this metabolic pathway can be used as a predictive tool for determining drug sensitivity. The following aims will be undertaken to achieve this goal. 1) Understand the mechanistic relationships among EGFR signaling, epithelial-to-mesenchymal transition (EMT) status, and GDRC. The effect of EGFR signaling on GDRC will be examined by knocking-down or inhibiting components of this pathway. Given that EMT has been linked to acquired resistance to EGFR inhibition (8-10), the impact of EGFR signaling on the epithelial phenotype of GDRC cells will also be analyzed. These studies will help determine whether GDRC will be useful as a predictive tool in determining drug sensitivity. 2) Identify additional shared vulnerabilities of GDRC cells based on perturbations of metabolism and signaling. Metabolomics data performed on high and low GDRC cells will be used to evaluate metabolic differences in GDRC cells and whether these can be exploited to target these cells. In addition, inhibitors targeting downstream EGFR signaling components, as well as other signaling pathways, will be used to identify other weaknesses shared by GDRC cells. 3) Examine the metabolic phenotypes and vulnerabilities associated with GDRC using an in vivo mouse xenograft model. High and low GDRC cell lines will be injected subcutaneously into the flanks of mice and the resulting tumors will be subjected to metabolic assays to analyze the GDRC phenotype. In addition, the impact of EGFR signaling on these tumors will be explored to determine whether GDRC would be useful as a predictive tool for drug sensitivity. Together, this work will provide insight into the regulation of GDRC metabolism in lung cancer and whether this pathway may prove useful as a predictive tool in cancer treatment.